Ab191866

The middle section of the graft was lysed in Radio Immunoprecipitation Assay (RIPA) buffer (Beyotime, Shanghai, China). BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, USA) was used to test the concentration of proteins. Total proteins (20 μg) were subjected to 6–10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to the polyvinylidene fluoride (PVDF) membranes. The membranes were cultured in 5% bovine serum albumin (BSA; Solarbio, Beijing, China) solution for 1 h and sequentially incubated with the primary antibodies overnight at 4°C, such as Skp2 (#4358), P27 (#3686), c-Myc (#5605), CyclinE1 (#20808) and GAPDH (#8884, all from Cell Signaling Technology, Danvers, USA). The membranes were washed three times with TBST for 5 min each time, and then the HRP-labelled secondary antibodies (ab191866, 1 mg/ml, Abcam, Cambridge, USA) were added and incubated for 1 h at room temperature. Specific signals were detected using the ECL western blotting detection system.

The 5-µm thick paraffin slices of myocardium were deparaffinized in an incubator (60˚C; 2 h) and rehydrated in a descending alcohol series before washing with distilled water. After antigen recovery, 3% H₂O₂ was used to eliminate the activity of endogenous peroxidase. The processed slices were blocked with 5% BSA for 30 min at room temperature before incubation with anti-TNF-α (1:200; cat. no. ab205587; Abcam) in a sealed wet box at 4˚C overnight, and then incubated with HRP-conjugated anti-rabbit secondary antibody (1:2,000; ab191866; Abcam) at room temperature for 1 h. After incubation with diaminobenzidine reagents (cat. no. DA1016; Beijing Solarbio Science & Technology Co., Ltd.) for color detection, the slices were counterstained with hematoxylin at room temperature for 3 min and images were captured under a light microscope (Eclipse TS100; Nikon Corporation).

Total proteins were extracted by RIPA. Proteins were separated by SDS-PAGE and electro-transferred onto PVDF membranes. The membranes were soaked in buffer containing 5% BSA for 1 h and then incubated with primary antibodies including anti‐p-mTOR (5536s, 1:1000, Cell Signaling Technology), anti‐mTOR (2983s, 1:1000, Cell Signaling Technology), anti‐p-PI3K (bs-6417R, 1:1000, Bioss), anti‐PI3K (bs-10657R, 1:1000, Bioss), anti‐p-AMPK (2531S, 1:1000, Cell Signaling Technology), anti‐AMPK (5832S, 1:1000, Cell Signaling Technology), anti‐p-AKT (Sc-293125, 1:1000, Santa), anti‐AKT (Sc-5298, 1:1000, Santa), anti‐PGC1α (bs-7535R, 1:1000, Bioss), anti‐LC3B (ab51520, 1:5000, Abcam), anti‐ZO-1 (61–7300, 1:1000, Invitrogen), anti‐Occludin (71–1500, 1:1000, Invitrogen), or anti‐Claudin-5 (34–1600, 1:1000, Invitrogen), at 4 °C overnight. Thereafter, the membranes were washed with Tris‐buffered saline Tween buffer and incubated with HRP‐rabbit (ab191866, 1:5000, Abcam) or HRP‐mouse (bs-0296G-HRP, 1:1000, Bioss) conjugated secondary antibody for 1 h at room temperature (RT). An Odyssey Infrared Imaging System was used to scan the membranes for further analysis. β-actin (CL594-66009, 1:1000, Proteintech) was used as loading control. Band intensities were quantified using Image J software.

Protein was extracted from cell lines for western blotting as previously described (19 (link)). The anti-Notch-1 antibody (1:200, ab52627; Abcam) was incubated overnight at 4°C. After washing with 0.1% PBST (0.1% Tween-20), membranes were probed with anti-mouse or anti-rabbit horseradish peroxidase secondary antibodies (1:100; cat. nos. ab131368 and ab191866; Abcam) at room temperature for 2 h. Enhanced Chemiluminescent reagent kit (GE Healthcare Life Sciences, Chalfont, UK) was applied to assist the staining. The blots were scanned by ChemiDoc™ XRS (Bio-Rad Laboratories, Hercules, CA, USA) and grey density was analyzed by Quantity One v4.62.

Western blot analysis was performed as previously described (23 (link)–28 (link)). Total protein was prepared using extraction buffer comprising NaCl/P_i containing 0.5% Triton X-100, 1 mM EDTA, 1 mM PMSF, and complete protease inhibitors (Roche Diagnostics, Basel, Switzerland). The concentration of each protein lysate was determined using a BCA™ protein assay kit (Thermo Fisher Scientific, Inc.). Equal quantities (20 µg) of total protein were subjected to 12% SDS-PAGE. The samples were then transferred onto nitrocellulose membranes and blocked for 60 min at room temperature in 5% skim milk powder in NaCl/P_i. The membranes were immunoblotted using antibodies against human LEFTY2 (ab204283; 1:500; Abcam, Cambridge, MA, USA), E-cadherin (ab40772; 1:500) and vimentin (ab92547; 1:500; all from Abcam), snail (ab82846; 1:500 Abcam) and β-actin (ab8227 1:500; Abcam) overnight at 4°C. IRDye^®-800 conjugated anti-rabbit secondary antibody (1:10,000; ab191866, Abcam) was used for incubation at room temperature for 30 min. The specific proteins were visualized using an Odyssey™ infrared imaging system (Gene Company, Ltd., Lincoln, NE, USA). The expression of β-actin was used as an internal control to ensure equal loading of the protein samples.

Western blotting was performed to measure the expression of Gli3, cleaved-caspase-3 and cleaved-PARP proteins. SiHa cells were homogenized in lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) and the total protein was extracted by centrifuged at 16,000 × g at 4°C for 5 min. The proteins (20 µg/lane) were quantified according to the protocol of the BCA kit (cat. no. AR0146; Wuhan Boshide Biological Engineering Co. Ltd., Wuhan, China), separated by 10% SDS-PAGE and transferred onto a polyvinylidene fluoride membrane. Membranes were subsequently incubated with primary antibodies against Gli3 (1:1,000, PA5-19822; Invitrogen, Thermo Fisher Scientific, Inc.), cleaved-caspase-3 (1:500; ab49822) cleaved-PARP (1:1,000; ab32064), cyclin B1 (1:500; ab72), cyclin D1 (1:10,000; ab134175; all Abcam, Cambridge, UK) and GAPDH (1:1,000 dilution; MA5-15738; Invitrogen; Thermo Fisher Scientific, Inc.) for 1 h at room temperature. GAPDH was the internal control. Membranes were then incubated with horseradish peroxidase-conjugated anti-mouse (1:1,000; ab131368) and anti-rabbit (1:1,000; ab191866; Abcam) antibodies at room temperature for 2 h. Finally, membranes were incubated with BeyoECL Plus (Beyotime Institute of Biotechnology) and the detected using Chemi Doc™ XRS+ imaging system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Cells were solubilized with radioimmunoprecipitation assay lysis buffer (Thermo Fisher Scientific, Inc.) and protein concentration was measured using a BCA kit (Thermo Fisher Scientific, Inc.). Approximately 50 µg of protein from each sample was separated by 12% SDS-PAGE and transferred to a polyvinylidene fluoride membrane (EMD Millipore, Burlington, MA, USA). Following blocking with 5% bovine serum albumin (1:100; Sigma-Aldrich; Merck KGaA) in Tris-buffered saline with Tween for 2 h at room temperature, membranes were incubated with Bcl-2 (ab178529), Bax (ab53154), pro-caspase-3 (ab32150) and active-caspase-3 (ab181418), phosphorylated (p)-p38 (ab4822), p38 (ab31828) and Ras (ab52939) primary antibodies at a dilution of 1:1,000 overnight at 4°C (all Abcam, Cambridge, UK), followed by incubation with goat anti-rabbit horseradish peroxidase conjugated secondary antibodies (at a dilution of 1:2,000; ab191866; Abcam) for 1 h at room temperature. GAPDH (ab8245; dilution 1:2,000; Abcam) was used as an internal control. Protein blots were visualized and analyzed using a chemiluminescence system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and autoradiography films (Kodak Image Station 440; Kodak, Rochester, NY, USA).